The present invention relates to application of recombinant DNA technology to plants. More specifically, the invention relates to the development of transgenic plants with enhanced chlorophyll content and enhanced salt tolerance.
Calcium plays an important role in cellular regulation in almost all organisms. Reference can be made to (Bootman, M D and Berridge, M J, 1995, The elemental principles of calcium signaling, Cell, 83, 675-678.) wherein the authors have described the importance of calcium signaling that acts as a secondary messenger through a series of calcium binding proteins. These proteins bind calcium and subsequently either directly or indirectly through another set of proteins, carry out different biological functions. Reference can be made to (Roberts, D M and Harmon, A C, 1992, Calcium modulated proteins: targets of intracellular calcium signals in higher plants, Ann. Rev. Plant Physiol. Plant Mol. Biology, 43, 375-414) wherein the authors have described various calcium modulated proteins including the calcium stimulated kinases and their involvement in various biological functions in plants. Calcium binding domains of many of these proteins have been characterized. Different calcium binding proteins have different number of calcium binding domains. Reference can be made to (Jang, H J, Pih, K T, Kang, S G, Lim, J H, Jim, J B, Piao, H L and Hwang, I, 1998, Molecular cloning of a novel calcium binding protein that is induced by NaCl stress, Plant Molecular Biology, 37, 839-847.) wherein the authors have described a novel salt stress induced calcium binding protein having three calcium binding loops as compared to 4 domains present in calmodulin. Though many calcium binding proteins have been reported in the literature, there are still a host of other proteins that have not yet been reported. Besides the biological role of many of these calcium binding proteins is not clear. Since calcium plays such an important role in biological systems it is important to characterise novel calcium binding proteins and decipher their role. A novel calcium binding protein from the protozoan parasite Entamoeba histolytica has recently been described by one of the applicants and regarding this, reference can be made to Prasad, J, Bhattacharya, S Bhattacharya, A, 1992, (Cloning and sequence analysis of a calcium binding protein gene from a pathogenic strain of Entamoeba histolytica. Mol. Biochem. Parasitol. 52, 137-140.), wherein the authors have described the properties of this novel EhCaBP and shown how this protein is different from well characterized calcium binding protein (CaBP), calmodulin. Nucleotide sequence comparison with the existing databases showed that it is a new kind of calcium binding protein not described so far.
Further, Prasad et al in Cellular and Molecular Biology Research, Vol. 39, pp.167-175, 1993 reported the expression of the EhCaBP in E.coli. and the use thereof to generate polyclonal antibodies . Nagendra Yadava et al in Molecular and Biochemical Parasitology 84 (1997) 69-82 reported the sub-cellular location of EhCaBP and its functional differences with CaM (Calmodulin).
The main objective of the invention is to modify the homologue(s), of the calcium binding protein gene in plants by recombinant DNA technology for enhanced chlorophyll production and retardation of leaf senescence in plants, and enhanced salt tolerance of seedlings.
Another object is to use of anti-sense and sense gene constructs of a calcium binding protein to manipulate expression of the said or similar genes. An object is to provide a method for expression of EhCaBP gene isolated from the protozoan parasite E. hystotitica under the control of a constitutive promoter.
Another object is to provide a method of expressing EhCaBP protein in plants using sense constructs of the said gene, under the control of a constitute promoter.
Yet another object is to develop anti-sense RNA constructs of the EhCaBP protein under the control of a constitute promoter for high level expression in different tissues of the plant.
A further object is to develop transgenic plants transformed with the antisense constructs of the EhCaBP gene and the constitutive promoter.
Accordingly, the invention provides methods for expression of EhCaBP gene isolated from E. hystolytica, under the control of a constitutive promoter, in plants. The invention also relates to the development of transgenic plants transformed with the antisense constructs of the EhCaBP gene and the constitutive promoter.
According to the present invention there is provided methods for enhanced chlorophyll production and enhanced salt tolerance of seedlings. Specifically the present invention relates to the use of anti-sense and sense gene constructs of a calcium binding protein to manipulate expression of the said or similar genes.
In accordance with the foregoing objects, the invention provides a nucleic acid construct comprising:
(a) an anti-sense gene of a sense gene encoding E. histolytica calcium binding protein or a portion of said anti-sense gene, wherein said sense gene is at least 90% similar to the nucleic sequence of SEQ ID No: 1, and wherein said portion of the anti-sense gene is of a size capable of disrupting translation of said calcium binding protein;
(b) a constitutive promoter and a nopaline synthase (nos) polyadenylation signal sequence both operatively linked to said gene or portion thereof, wherein said construct is useful for increasing the level of chlorophyll in plants.
In an -embodiment, the promoter is selected from the group of commercially available promoters comprising CaMV 35S, actin and ubiquitin.
In another embodiment, the preferred promoter is CaMV 35S.
In another embodiment, the antisense gene is at least 90% similar to the nucleic acid sequence of SEQ ID NO:1.
Another embodiment provides a transgenic plant containing a nucleic acid sequence of SEQ ID NO:1.
Further, the invention provides a nucleic acid construct comprising.
(a) a sense gene encoding E. histolytica calcium binding or an altered sense gene wherein said gene encodes proteins of a sequence having biological properties identical to the said sense gene;
(b) a constitutive promoter and a nopaline syntheses (nos) polyadenylation signal sequence both operatively linked to said gene or portion thereof, wherein the said construct is useful in developing stress-tolerant seed plant.
In another embodiment, the promoter is selected from the group of commercially available promoters comprising CaMV 35S, actin and ubiquitin.
In embodiment, the preferred promoter is CaMV 35S.
In yet another embodiment, the antisense gene is at least 95% similar to the nucleic acid sequence of SEQ ID NO.1.
The invention also provides transgenic plants containing a nucleic acid sequence of SEQ ID NO.1.
Further, the invention relates to a method for increasing chlorophyll content in plants, said method comprising the steps of:
a) preparing the nucleic acid construct or sequence of SEQ ID NO.1 capable of manipulation of calcium binding protein, and comprising the gene encoding E. histolytica calcium binding protein; and
b) transforming a plant with the said construct.
In an embodiment is provided a method for increasing salt tolerance of plants, comprising the steps of:
a) preparing the nucleic acid construct or sequence of SEQ ID NO.1. capable of increasing the concentration of E. histolytica calcium binding protein, and comprising the gene encoding E. histolytica calcium binding protein; and
b) transforming a plant with the said construct.
In one embodiment there is provided a method of using anti-sense E. histolytica calcium binding protein (EhCaBP) gene.
The anti-sense constructs of the present invention are under the control of a constitutive promoter, such that chlorophyll accumulation is observed throughout the development of the plants.
Thus, according to one embodiment of the present invention there is provided an anti-sense construct comprising an anti-sense RNA sequence for the E. histolytica calcium binding protein (EhCaBP) gene and a constitutive promoter.
In yet further embodiment of the present invention there is provided a transgenic plant comprising an anti-sense RNA sequence for the EhCaBP gene and a constitutive promoter.
In another embodiment of the present invention there is provided a method of expressing EhCaBP protein using sense constructs of the said gene.
The sense constructs of the present invention are under the control of CaMV 35S, a constitutive promoter, such that the transgenic plants express protein and become tolerant to low salinity.
Thus, according to one embodiment of the present invention there is provided an antisense construct comprising a sense RNA sequence for the EhCaBP gene and constitutive promoter.
In yet further embodiment of the present invention there is provided a transgenic plant comprising a sense RNA sequence for the EhCaBP gene and a constitutive promoter.